Method for template-based enzymatic DNA synthesis using phosphoryl guanidine oligonucleotides and reaction mixtures for carrying out the method

Abstract

The invention relates to the development and optimization of PCR and RT-PCR systems used to detect nucleic acids, including the diagnosis of genetic, viral, and other diseases. The essence of the proposed method is that neutral derivatives of oligonucleotides, namely phosphoryl guanidines containing one or more phosphate groups in which guanidine or substituted guanidine residue is introduced on the phosphorus atom, are used as primers for the template-based amplification, including polymerase chain reaction (PCR) and PCR combined with reverse transcription (RT-PCR). The invention allows to obtain more reliable, specific and selective results in the process of PCR, in particular, to increase the sensitivity of PCR by reducing the yield of by-products of DNA amplification and/or to control the yield of the PCR product, including intentionally suppressing, by using different combinations of the location and number of modified phosphate groups in the oligonucleotide primers.

We claim: 
     
       1. A method for template-based enzymatic DNA synthesis, wherein a primer containing at least one phosphoryl guanidine group corresponding to the General formula (I): 
       
         
           
           
               
               
           
         
         where, 
         each X and Y is independently selected from nucleoside or oligonucleotide; and 
         each of the substituents R1, R2, R3, and R4 can be hydrogen atom H or C 1 -C 5  alkyl, or any two from R1-R4, together to form C 2 -C 5  alkanediyl, is used to initiate the reaction. 
       
     
     
       2. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used for amplification of nucleic acids. 
     
     
       3. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used for carrying out polymerase chain reaction. 
     
     
       4. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used for carrying out allele-specific polymerase chain reaction. 
     
     
       5. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used for reverse transcription. 
     
     
       6. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used in both stages of reverse transcription and subsequent polymerase chain reaction. 
     
     
       7. The method for template-based enzymatic DNA synthesis according to  claim 1 , which method is used for amplification of nucleic acids by the rolling circle mechanism. 
     
     
       8. A reaction mixture for template-based enzymatic DNA synthesis according to  claim 1  further comprising a primer containing at least one internucleotide phosphoryl guanidine group corresponding to the formula (I). 
     
     
       9. A set of reaction mixtures for template-based enzymatic DNA synthesis according to  claim 1  further comprising a primer containing at least one internucleotide phosphoryl guanidine group corresponding to the formula (I).